It is well known that anaerobic bacteria grow selectively in tumors, and indeed, bacteria in large numbers have been found in tumors excised from patients. In view of this, bacteria have been proposed as tumor therapeutics. For example, Yazawa, K., et al., Breast Canc. Res. and Treat. (2001) 66:165-170 demonstrated that Bifidobacterium longum, a non-pathogenic Gram-position bacterium, when injected intravenously, grew selectively in mammary tumors induced in rats. Dang, L. H., et al., Proc. Natl. Acad. Sci. USA (2001) 98:15155-15160 demonstrated that a strain of Clostridium novyi which had been depleted of its lethal toxin produced spores that germinated within avascular regions of tumors in mice and destroyed surrounding viable tumor cells.
Salmonella typhimurium, which are auxotrophic by virtue of disruption of the aro gene, were shown to have suppressed virulence and thus useful as live vaccines as described by Hoiseth, S. K. J., et al., Nature (1981) 291:238-239. The disruption of the aro gene blocked the production of both para-aminobenzoic acid (PABA) and of 2,3-dihydroxybenzoate (DHB). Earlier work that resulted in Salmonella auxotrophic only for PABA, and Salmonella auxotrophic only for DHB also resulted in reduced virulence, but disruption of the aro gene, resulting in the double auxotroph were characterized as virtually non-virulent. Pawelek, J., et al., Cancer Res. (1997) 57:4537-4544 describe Salmonella mutants that were pur (requiring adenine and vitamin B1) as antitumor therapeutics with reduced pathogenicity. Low, B., et al., Nature Biotech. (1999) 17:37-41 reported lipid-A-mutant Salmonella with disruption in the msbB gene which reduced TNFα induction and increased LD50 by 10,000-fold. Tumor accumulation ratios in excess of 1,000:1 compared with normal tissues were reported. Melanomas in mice treated with these modified Salmonella were 6% of the size of tumors in untreated controls.
Further, an attenuated S. typhimurium has been evaluated in a Phase I clinical trial, as reported in Toso, J. F., et al., J. Clin. Oncol. (2002 20:142-152. Twenty-four patients with metastatic melanoma and one patient with metastatic renal cell carcinoma received IV bolus infusions containing 106 to 109 cfu/m2 of the lipid-A-mutant Salmonella typhimurium (VNP20009) that was attenuated by deletion of both the purI and msbB genes. The purI mutant requires an external source of adenine, and the msbB mutant prevents the addition of a terminal myristyl group to the lipid-A domain. The VNP20009 strain could be safely administered to patients, and at the highest tolerated dose, tumor colonization was observed.
The precision of bacterial treatment of tumors could, of course, be enhanced by systems for tracking the progress of bacterial infection. One such system is described in U.S. patent publication 2003-0161788, published 28 Aug. 2003 and incorporated herein by reference. As described in this publication, bacteria such as E. coli, labeled with fluorescent proteins are used to monitor infection and to evaluate treatments. In model systems, and indeed, in treated subjects, in addition to direct observation of the labeled bacteria per se, tumor tissue may be provided with a fluorescent label of a different color to provide a desirable contrast. In the exemplified embodiments, S. typhimurium modified to express a variant of the A. victoria green fluorescent protein (GFP) were injected into tumors labeled with red fluorescent proteins (RFP's) in nude mice. In alternative embodiments, E. coli modified to express RFP were injected into nude mice containing tumors labeled with GFP. It was noted that in addition to their own inherent antitumor affects, the bacteria also might be modified to produce a therapeutic, such as IL2 or methioninase.
Yu, Y. A., et al., Nat. Biotechnol. (2004) 22:313-320 have shown that bacteria, expressing GFP, injected intravenously into live animals replicate in solid tumors and metastases including tumors of the breast, prostate, brain and fibrosarcoma. Growth in tumors was imaged in real time using luciferase-catalyzed luminescence as well as GFP. E. coli and three attenuated pathogens, Vibrio cholerae, S. typhimurium, and Listeria monocytogenes, replicated in tumors.
There remains a need for improved bacterial strains with enhanced selectivity for tumors and reduced toxicity such that these improved strains may be used as therapeutics, vaccines, and as guides to monitoring tumor progression and evaluating and optimizing tumor treatment. The present invention provides such improved strains of bacteria. An abstract describing the effects of S. typhimurium, prepared in accordance with the invention, but not describing the nature of the mutations, was published by Zhao, M., et al., Proc. Am. Assoc. Cancer Res. (2004) 45:869 (No. 3765).
Detailed description of the auxotrophic strain A1 described hereinbelow is set forth in Zhao, M., et al., Proc. Natl. Acad. Sci. USA (2005) 102:755-760, incorporated herein by reference. A short summary appears in Nature Biotechnology (2005) 23:189.